Peptide, process for preparation thereof and use thereof

ABSTRACT

The invention deals with novel peptides useful for the therapeutic treatment of infectious diseases caused by pathogenic microorganisms, having the structure ##STR1## wherein R 1  is substituted or unsubstituted lower alkanoyl, or substituted or unsubstituted aralkanoyl, 
     R 2  and R 3  are each carboxy or protected carboxy, 
     R 4  is hydrogen or an amino protective group, and 
     R 5  is hydrogen or lower alkyl, and their pharmaceutically acceptable salts.

This invention relates to a new peptide. More particularly, thisinvention relates to a new peptide and the pharmaceutically acceptablesalt thereof, which have pharmacological activities, to processes forthe preparation thereof and to a new intermediate for preparing theactive peptide, and to the pharmaceutical composition comprising thesame and a method of use thereof.

A new peptide of this invention is represented by the following formula(I): ##STR2## wherein R¹ is substituted or unsubstituted lower alkanoyl,or substituted or unsubstituted aralkanoyl,

R² and R³ are each carboxy or protected carboxy,

R⁴ is hydrogen or an amino protective group, and

R⁵ is hydrogen or lower alkyl.

Particulars of the various definitions, which are mentioned hereinaboveand hereinafter, and preferred examples thereof are explained in thefollowing.

The term "lower" is intended to mean a group having 1 to 8 carbon atoms,unless otherwise provided.

(1) Re. Substituted or unsubstituted lower alkanoyl for R¹ :

As suitable examples of unsubstituted lower alkanoyl, there may beexemplified formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl,isovaleryl, pivaloyl, hexanoyl, α-ethylhexanoyl, heptanoyl, octanoyl andthe like.

As substituted lower alkanoyl, there may be exemplified lower alkanoylas illustrated above which is substituted by one or more suitablesubstituent(s) such as amino, halogen (e.g. fluorine, chlorine, bromine,etc.), hydroxy, carboxy and the like, and among substituted loweralkanoyl, there may be exemplified hydroxy (lower) alkanoyl such as2-hydroxypropionyl (i.e. lactoyl) as more suitable example.

In the above exemplified substituted lower alkanoyl in case that theselower alkanoyl has one or more functional group(s) such as hydroxy,amino and carboxy, such a functional group may be protected by aconventional protective group.

(2) Re. Substituted or unsubstituted aralkanoyl for R¹ :

As a suitable unsubstituted aralkanoyl, there may be exemplifiedar(lower) alkanoyl such as mono or di-phenyl(lower)alkanoyl (e.g.phenylacetyl, diphenylacetyl, etc.) and the like.

As a suitable substituted aralkanoyl, there may be exemplifiedar(lower)alkanoyl, arene and (or) alkane moiety of which is substitutedby one or more suitable substituent(s) such as the same as thoseexemplified as the suitable substituent for substituted lower alkanoylfor R¹.

Among said substituted aralkanoyl, as suitable example there may beexemplified ar(lower)hydroxyalkanoyl such as mandelyl and the like.

In the above exemplified substituted aralkanoyl, in case that thesearalkanoyl has one or more functional group such as hydroxy, amino andcarboxy, such a functional group may be protected by a conventionalprotective group.

(3) Re. Protected carboxy for R², R³, and R_(a) ³, and functional groupin the group for R¹ :

A protective group of the protected carboxy includes a conventionalcarboxy protective group which is conventionally used in the field ofamino acid and peptide chemistry.

As suitable examples of protected carboxy, there may be exemplified anester such as an ester with silyl compound, an ester with an aliphatichydroxy compound and an ester with a hydroxy compound containing anaromatic group.

As more suitable examples of protected carboxy, there may be exemplifiedaralkyl (e.g. benzyl, diphenylmethyl, etc.) ester and the like.

(4) Re. Amino protective group for R⁴, R_(a) ⁴ and R_(b) ⁴, andfunctional group in the group for R¹ :

The amino protective group includes a conventional amino protectivegroup which is used in the field of amino acid and peptide chemistry.

As suitable examples of the amino protective group, there may beexemplified alkoxycarbonyl (e.g. methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, t-pentoxycarbonyl,etc.), aralkoxycarbonyl (e.g. benzyloxycarbonyl, etc.) and the like.

(5) Re. Hydroxy protective group in the group for R¹ :

As suitable example of a hydroxy protective group in substituted loweralkanoyl and substituted aralkanoyl for R¹, there may be exemplified aconventional one, for example, acyl such as alkanoyl (e.g. acetyl,etc.).

(6) Re. Lower alkyl for R⁵ :

Suitable example of lower alkyl for R⁵ is one having 1 to 6 carbon atomssuch as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl,isopentyl and the like.

A pharmaceutically acceptable salt of the new peptides of the formula(I) may include a salt with an inorganic or organic base such as analkali metal salt (e.g. sodium salt, potassium salt, etc.), an alkalineearth metal salt (e.g. calcium salt, etc.), ammonium salt, organic aminesalt (e.g. ethanolamine salt, triethylamine salt, dicyclohexylaminesalt, etc.) or the like, and an acid addition salt with organic orinorganic acid such as methane sulfonate, hydrochloride, sulfate,nitrate, phosphate or the like. The compound (I) of this invention canbe prepared by various methods, details of which will be apparent fromthe following descriptions. ##STR3##

In the above formula, R¹, R³ and R⁵ are each as defined above, R_(a) ²is protected carboxy, and R_(a) ⁴ is amino protective group.

Detailed explanation of processes for preparing of the compound (I) willbe made in the following:

(1) Process 1: Peptide bond formation Compound (II)+Compound(III)→Compound (Ia)

This process relates to a method for preparing Compound (Ia) by reactingCompound (II) or its salt with a Compound (III) or its salt.

The reaction of this process can be conducted as follows.

That is, in one case, as the first step, the carboxy group of Compound(II) or its salt is usually activated in a conventional manner, forexample, in the form of its acid halide, azide, acid anhydride or amixed anhydride, activated ester, and the like, and is reacted with theCompound (III) to give Compound (Ia), and in the other case, theCompound (II) or its salt is reacted with the Compound (III) or its saltdirectly in the presence of a conventional condensing agent such asN,N-dicyclohexylcarbodiimide and the like. Among these activationmethods, preferred activation method for the carboxy group of theCompound (II) into its activated form and preferred condensing agent asmentioned above are selected according to kinds of the carboxyprotective group(s) of the Compound (II) and (III) and to the reactionconditions (e.g. the kinds of the reaction solvent, reaction temperatureand so on).

This reaction is preferably carried out in a solvent such as methylenechloride, chloroform, tetrahydrofuran, dioxane, ethyl acetate, methanol,ethanol, water or the like under at -20° C. to at ambient temperatureand the reaction in the presence of a condensing agent is usuallycarried out in an anhydrous, but not critical conditions.

(2) Process 2: Elimination of protective group(s) Compound(I^(a))→Compound (I^(b))

This process relates to a method for preparing Compound (I^(b)) bysubjecting Compound (I^(a)) to elimination reaction of protectivegroup(s) of protected carboxy for R_(a) ² and (or) R³, and (or) an aminoprotective group for R⁴, detailed explanation for which is as follows:

Process 2-1: Elimination of an amino protective group for R⁴

This process can be applied to case that the amino protective group forR⁴ reveals a chemically different behavior from that of the acyl groupfor R¹ against each kind of the elimination methods to be employed, thatis, the case that the amino protective group can be eliminated, but theacyl group for R¹ is not eliminated according to the elimination methodas employed.

This reaction is carried out by conventional methods such as catalyticreduction method, liquidammoniaalkalimetal method, acid method, zincacid method, base method, hydrazine method and the like.

Among these methods, preferred one is selected according to kind of theamino protective group for R⁴, and also to the chemically differentbehavior of said amino protective group from the acyl for R¹ asexplained above.

Among the above elimination methods, an acid method is employed as themost convenient and conventional one and said method is explained asfollows:

This reaction is conventionally carried out in a solvent such asmethylene chloride, chloroform, acetic acid, water and the like in thepresence of inorganic or organic acid such as trifluoroacetic acid,formic acid, p-toluenesulfonic acid, hydrochloric acid, hydrobromic acidand the like and anisole is preferably added thereto.

Among the exemplified acid, trifluoroacetic acid and formic acid arealso used as the solvent.

This reaction is usually carried out under ice-cooling to an ambienttemperature.

Process 2-2: Elimination of carboxy protective group of protectedcarboxy for R_(a) ² and R³

The reaction for elimination of protective group of the protectedcarboxy group is carried out by a conventional method such as hydrolysisand reduction or the like, details of which are explained in thefollowing.

(i) For hydrolysis which refers to the same meaning as solvolysisincluding, for example, acidolysis, alcoholysis, aminolysis,hydroxinolysis, etc.:

Hydrolysis is preferably carried out in the presence of an acid or base.

Suitable acid includes an inorganic acid (e.g. hydrochloric acid,hydrobromic acid, sulfuric acid, etc.), an organic acid (e.g. formicacid, acetic acid, trifloroacetic acid, propionic acid, benzenesulfonicacid, p-toluenesulfonic acid, etc.), an acidic ion exchange resin andthe like.

Suitable base includes an inorganic base such as alkali or alkalineearth metal hydroxide or the corresponding carbonate or bicarbonate(e.g. sodium hydroxide, potassium hydroxide, sodium carbonate, potassiumcarbonate, lithium carbonate, sodium bicarbonate, calcium hydroxide,magnesium hydroxide, etc.), ammonium hydroxide or the like; an organicbase such as an alkoxide or phenoxide of the above metal (e.g. sodiumethoxide, sodium methoxide, lithium phenoxide, etc.), an amine such asmono-, di- or trialkylamine (e.g. methylamine, ethylamine, propylamine,isopropylamine, butylamine, N,N-dimethyl-1,3-propanediamine,trimethylamine, triethylamine, etc.), unsubstituted, mono- ordisubstituted arylamine (e.g. aniline, N-methylaniline,N,N-dimethylaniline, etc.), a heterocyclic base (e.g. pyrrolidine,morpholine, N-methylmorpholine, N-methylpiperidine,N,N-dimethylpiperazine, pyridine, etc.), hydrazines (e.g. hydrazine,methylhydrazine, ethylhydrazine, etc.) or the like; a basic ion-exchangeresin and the like.

The hydrolysis is preferably conducted under somewhat milder conditionssuch as cooling or warming and usually in a solvent which does not haveadverse influence to the reaction, e.g. water, a hydrophilic solventsuch as alcohol (e.g. methanol, ethanol, propanol, etc.), acetone,N,N-dimethylformamide, tetrahydrofuran, dioxane, dimethylsulfoxide, etc.or a mixture thereof, and other hydrophobic solvent such as benzenediethylether, etc. may also be used as a solvent. A liquidabovementioned acid or base can also be used as solvent.

(ii) For reduction:

Reduction, including chemical reduction and catalytic reduction, iscarried out in a conventional manner.

Suitable reducing agents to be used in chemical reduction are a metal(e.g. tin, zinc, iron, etc.), or a combination of such metal and/ormetallic compound (e.g. chromium chloride, chromium acetate, etc.) andan organic or inorganic acid (e.g. formic acid, acetic acid, propionicacid, trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid,hydrobromic acid, etc.).

Suitable catalysts to be used in catalytic reduction are conventionalones such as platinum catalysts (e.g. platinum plate, spongy platinumplatinum black, colloidal platinum, platinum oxide or platinum wire,etc.), palladium catalysts (e.g. spongy palladium, palladium black,palladium oxide, palladium on carbon, colloidal palladium, palladium onbarium sulfate, palladium on barium carbonate, etc.), nickel catalysts(e.g. reduced nickel, nickel oxide, Raney nickel, etc.), cobaltcatalysts (e.g. reduced cobalt, Raney cobalt, etc.), iron catalysts(e.g. reduced iron, Raney iron, etc.), copper catalysts (e.g. reducedcopper, Raney copper, Ullman copper, etc.) or the like.

The reduction is usually carried out in a solvent. A suitable solvent tobe used may be, e.g. water, alcohol (e.g. methanol, ethanol, propanol,etc.) and other conventional organic solvent or a mixture thereof.Additionally, the afore-mentioned liquid acids to be used in chemicalreduction can also be used as solvent. Further, a suitable solvent to beused in catalytic reduction may be, e.g. the above-mentioned solvent,and other conventional solvent, such as diethyl ether, dioxane,tetrahydrofuran, etc. or a mixture thereof.

The reaction is preferably carried out under somewhat milder conditionssuch as cooling or warming.

Among these methods for elimination of protective groups, preferred oneand appropriate combination methods are to be selected according tokinds of the protective groups of carboxy group and amino protectivegroup to be removed off.

It is noted that this process includes the following cases ofelimination of protective groups of protected carboxy and aminoprotective group, that is, one case that all of the carboxy protectivegroups for R_(a) ² and R³ and the amino protective group for R⁴ in theCompound (I^(a)) are simultaneously removed by a method to be employedto the reaction, and the other case that the carboxy protective groupsand the amino protective group are sequentially and stepwise removed bya method which is appropriately selected according to the kinds of theprotective group to be removed.

As to Process 2 for elimination of protective group(s) (i.e. Process 2-1and 2-2), the followings are to be noted. That is, in case thatsubstituted or unsubstituted lower alkanoyl, or substituted orunsubstituted aralkanoyl group for R¹ has one or more protectivegroup(s) for hydroxy, amino and (or) carboxy, such an amino protectivegroup and carboxy protective group among said protective group may besimultaneously removed in this process, and such a hydroxy protectivegroup such as alkanoyl (e.g. acetyl, etc.) may be previously removed bysubjecting the compound (I^(a)) to elimination reaction of hydroxyprotective group in a conventional manner such as reduction asillustrated in the Process 2-2.

The starting compounds (II) and (III) can be prepared by methods asfollows: ##STR4##

In the above formulae, R_(b) ⁴ is an amino protective group, and R_(a)², R_(a) ⁴ and R⁵ are as defined above. (1) Process 1^(s) : Compound(III-1)→Compound (III-2)

This process relates to a method for preparing compound (III-2) bysubjecting Compound (III-1) to amidation reaction.

The reaction is usually carried out as the first step by activating thecarboxy group of the compound (III-1) in a conventional manner, forexample, in a form of its activated ester, and then reacting theresulting compound with ammonia.

This reaction is preferably carried out in a solvent such as methylenechloride, chloroform or the like under ice-cooling to at ambienttemperature.

(2) Process 2^(s) : Compound (III-2)→Compound (III-3)

This process relates to a method for preparing compound (III-3) bysubjecting compound (III-2) to elimination reaction of protective groupof protected carboxy for R_(a) ².

This reaction is carried out substantially in the same manner as that ofProcess 2-2.

As to the object compound (I) and starting compounds (II) and (III)which are prepared according to the aforementioned Processes, it is tobe noted that each of said compounds includes one or more stereoisomerswhich is due to the asymmetric carbon atoms in their molecule and all ofsuch isomers are included within the scope of this invention.

The new peptide (I) and its pharmaceutically acceptable salts of thisinvention have been found to possess protective efficacy in experimentalinfection.

Accordingly, the new peptide (I) and its pharmaceutically acceptablesalts are useful for the therapeutic treatment of infectious diseasescaused by pathogenic microorganism, especially gram-negative bacteriaand gram-positive bacteria and fungi.

Further, Compounds (II) and (III) are useful as intermediate forpreparing Compound (I) having biologically active properties asmentioned above.

For the purpose of showing pharmaceutical utility of the new peptide(I), pharmacological test data thereof are illustrated in the following.

PROTECTIVE EFFICACY IN EXPERIMENTAL INFECTION IN MICE

In determining the protective efficacy against experimental infectionsin mice, the test compound was dissolved in and diluted with sterilesaline to provide prescribed concentrations of drug.

Male ICR-strain mice, aged 4 weeks were used in groups of ten mice. E.coli 22 was cultivated overnight at 37° C. on trypticase soy agar andthen were suspended in a sterile saline to obtain microbial cellconcentration of 2.6×10⁹ CFU/ml. Mice were inoculated intraperitoneallywith 8.7×10⁷ CFU/mouse. Each of the test drugs was givenintraperitoneally in various doses to a group of ten mice four daysbefore challenge.

Survival percent were found from the number of the surviving animalsafter three days of injection. Results are shown in Table

    ______________________________________                                                  Survival (%)                                                        Test Compound                                                                             Dose        Dose      Dose                                        (Example NO.)                                                                             0.1 mg/kg   0.01 mg/kg                                                                               0                                          ______________________________________                                        Example 1   50          100       10                                          (Step 2)                                                                      Example 2   80          30        30                                          (Step 2)                                                                      Example 3   90          70        30                                          (Step 2)                                                                      ______________________________________                                    

The pharmaceutical composition of this invention can be used in the formof a pharmaceutical preparation, for example, in solid, semisolid orliquid form, which contains an active substance of this invention inadmixture with an organic or inorganic carrier or excipient suitable forexternal, enteral or parenteral applications. The active ingredient maybe compounded, for example, with the usual non-toxic, pharmaceuticallyacceptable carriers for tablets, pellets, capsules, suppositories,solutions, emulsions, suspensions, and any other form suitable for use.The carriers which can be used are water, glucose, lactose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, collidal silica, potato starch, urea and other carrierssuitable for use in manufacturing preparations, in solid, semisolid, orliquid form, and in addition auxiliary, stabilizing, thickening andcoloring agents and perfumes may be used. The pharmaceuticalcompositions can also contain preservative or bacteriostatic agents tokeep the active ingredient in the desired preparations stable inactivity. The active object compound is included in the pharmaceuticalcomposition in an amount sufficient to produce the desired therapeuticeffect upon the process or condition of diseases.

For applying this composition to humans, it is preferably to apply it byintravenous, intramuscular or oral administration. While the dosage ortherapeutically effective amount of the object compound of thisinvention varies from and also depends upon the age and condition ofeach individual patient to be treated, a daily dose of about 0.1-100 mgof the active ingredient/kg of a human being or an animal is generallygiven for treating diseases, and an average single dose of about 50 mg,100 mg, 250 mg, and 500 mg is generally administered.

The following examples are given for purpose of illustrating thisinvention.

In the following examples, starting compounds and object compounds areexpressed by using the following abbreviations:

Lac: Lactoyl

Ala: Alanyl

Glu: Glutamyl

Gly: Glycyl

DAP: α, ε-Diaminopimelyl

Z: benzyloxycarbonyl

Boc: t-butoxycarbonyl

Bzl: benzyl

Ac: acetyl

Preparation 1 ##STR5##

To a mixture of Z-(L)-Boc-(D)-mesoDAP-(L)-GlyOBzl (1)(3.95 g) andN-methylmorphorine (0.85 g) in dry methylene chloride (70 ml) was addedisobutyl chloroformate (0.95 g) at -10°˜-15° C. and the mixture wasstirred for 30 minutes at the same temperature. The mixture was thencooled to -40° C. and 2N solution (15 ml) of ammonia in ethanol wasadded. After stirring for 30 minutes at the same temperature, themixture was concentrated and the residue was dissolved in ethyl acetate,washed with water, dried over magnesium sulfate, and evaporated to givean oil, which was pulverized with ether to giveZ-(L)-Boc-(D)-mesoDAP-(D)-NH₂ -(L)-GlyOBzl (2)(3.1 g).

IR (Nujol): 3300, 1735, 1685, 1655 cm⁻¹.

NMR (CD₃ OD): δ1.48 (9H, s), 1.4-2.0 (6H, m), 4.03 (2H, s), 3.9-4.3 (2H,m), 4.13 (2H, s), 4.22 (2H, s), 7.40 (10H, s).

Preparation 2 ##STR6##

To a solution of Z-(L)-Boc-(D)-mesoDAP-(D)-NH₂ -(L)-GlyOBzl (1)(2.7 g)in a mixture of methanol (75 ml) and water (10 ml) was added 10%palladium-charcoal (1.0 g) and the mixture was hydrogenated under anatmospheric pressure of hydrogen. After removal of the catalyst, thefiltrate was evaporated to dryness. The residue was pulverized withether to give Boc-(D)-mesoDAP-(D)-NH₂ -(L)-GlyOH (2)(1.59 g).

IR (Nujol): 3600-2200, 1690 (sh), 1670 cm⁻¹.

NMR (CD₃ OD): δ1.42 (9H, s), 1.2-2.0 (6H, m), 3.6-4.1 (4H, m).

EXAMPLE 1 (1) Step (1) ##STR7##

A mixture of D-Lac(oAc)-L-Ala-D-Glu(OH)OBzl (1) (1.15 g) andN-methylmorpholine (0.27 g) in methylene chloride (55 ml) was cooled to-15° C. and isobutyl chloroformate (0.37 g) was added dropwise. Afterstirring for 30 minutes at -10°˜15° C., the mixture was cooled to -40°C. and a solution of Boc-(D)-mesoDAP-(D)-NH₂ -(L)-GlyOH (2)(0.94 g) andbis(trimethylsilyl)acetamide (2.76 g) in methylene chloride (25 ml) wasadded. The mixture was stirred for 1.5 hours at -10°˜15° C. and for 1.5hours at -15°˜0° C. The mixture was concentrated and the residue wasdissolved in ethyl acetate (80 ml). The solution was washed successivelywith 2.5% hydrochloric acid and brine, dried over magnesium sulfate andevaporated to give a crystalline solid, which was washed with ether togive D-Lac(oAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NH.sub.2-(L)-GlyOH (3)(1.80 g).

IR (Nujol): 3280, 1730, 1655, 1640 cm⁻¹.

NMR (CD₃ OD): δ1.2-2.2 (25H, m), 2.12 (3H, s), 3.97 (2H, s), 4.2-5.0(5H, m), 5.20 (2H, s), 7.39 (5H, s).

(2) Step 2 ##STR8##

A solution of D-Lac-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NH₂-(L)-GlyOH (3)(1.67 g) in a mixture of methanol (50 ml) and water (8 ml)was hydrogenated over 10% palladium-charcoal (1.0 g) under anatmospheric pressure of hydrogen. After removal of the catalyst, thefiltrate was evaporated to dryness and the residue was dissolved intrifluoroacetic acid (15 ml) and stirred for 30 minutes at roomtemperature. After evaporation of trifluoroacetic acid, the residue wasdissolved in 50% aqueous methanol (44 ml) and adjusted to pH 9 with 10%sodium carbonate. After stirring for 1 hour at room temperature, themixture was concentrated and the resulting aqueous solution was adjustedto pH 2 with 5% hydrochloric acid and put on a column of an activatedcharcoal (30 ml). The column was washed with water and eluted with 80%aqueous methanol. The eluate was concentrated and the residue waschromatographed on a column of a macroporous non-ionic adsorption resin,HP-20 (230 ml), eluting with water. The eluate was concentrated andlyophilized to give D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-NH₂-(L)-GlyOH (4)(0.81 g).

[α]_(D) -34.4° (C=0.2, H₂ O).

IR (Nujol): 3280, 1720 (sh), 1650 cm⁻¹.

NMR (D₂ O): δ1.38 (3H, d, J=7 Hz), 1.42 (3H, d, J=7 Hz), 1.6-2.5 (10H,m), 3.96 (2H, s), 4.03 (1H, t, J=6 Hz), 4.2-4.5 (5H, m).

EXAMPLE 2 (1) Step 1

Heptanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NH.sub.2-(L)-D-AlaOH was prepared substantially in the same manner as step 1 ofExample 1.

NMR (CD₃ OD), δ: 0.93 (3H, t, J=5 Hz), 1.1-2.5 (35H, m), 3.9-4.6 (5H,m), 5.17 (2H, s), 7.35 (5H, s).

IR (Nujol) cm⁻¹ : 3260, 1720, 1630, 1520, 1330, 1290, 1265, 1165.

(2) Step 2

Heptanoyl-L-Ala-γ-D-GLu(α-OH)-(L)-mesoDAP-(D)-NH₂ -(L)-D-AlaOH wasprepared substantially in the same manner as step 2 of Example 1.

NMR (D₂ O), δ: 0.87 (3H, t, J=5 Hz), 1.37 (3H, d, J=7 Hz), 1.40 (3H, d,J=7 Hz), 1.1-2.5 (20H, m), 3.9-4.5 (5H, m).

IR (Nujol) cm³¹ 1 : 3280, 1690 (sh), 1640, 1540, 1230, 1165.

EXAMPLE 3 (1) Step 1

Phenylacetyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NH.sub.2-(L)-D-AlaOH was prepared substantially in the same manner as step 1 ofExample 1.

NMR (CD₃ OD), δ: 1.2-2.4 (25H, m), 3.56 (2H, s), 4.1-4.5 (5H, m), 5.14(2H, s), 7.28 (5H, s), 7.34 (5H, s).

IR (Nujol) cm⁻¹ : 3250, 1720, 1660, 1625, 1520, 1290, 1240, 1205, 1160.

(2) Step 2

Phenylacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-NH₂ -(L)-D-AlaOH wasprepared substantially in the same manner as step 2 of Example 1.

NMR (D₂ O), δ: 1.32 (3H, d, J=6 Hz), 1.38 (3H, d, J=6 Hz), 1.4-2.2 (10H,m), 3.65 (2H, s), 3.8-4.5 (4H, m), 7.33 (5H, s).

IR (Nujol) cm⁻¹ : 3270, 1640, 1530, 1240, 1160, 1025.

EXAMPLE 4 (1) Step 1 ##STR9##

Isobutyl chloroformate (114 mg) was added to a mixture ofD-Lac(OAc)-L-Ala-D-Glu(α-OBzl)OH (1) (351 mg) and triethylamine (84 mg)in methylene chloride (17 ml) at -10° to -15° C. and the mixture wasstirred for 30 minutes at the same temperature. The mixture was thencooled to -40° C. and Z-(L)-mesoDAP-(D)-NH₂ -(L)-GlyOH(2) trimethylsilylester, prepared from Z-(L)-mesoDAP-(D)-NH₂ -(L)-GlyOH (316 mg) andbis(triemthylsilyl)acetamide (1.4 ml) by stirring in methylene chloride(8.5 ml), was added and the mixture was stirred for 5.5 hours at -10° to-15° C. After evaporation of the solvent, the residue was dissolved inethyl acetate (80 ml) and washed with 2% hydrochloric acid (20 ml) andwater (10 ml). Ethyl acetate was evaporated to dryness and the residuewas pulverized with ether to giveD-Lac(OAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Z-(D)-mesoDAP-(L)-GlyOH-(D)-NH₂ (3)(457 mg). mp 172°-178° C. (dec).

IR (Nujol) cm⁻¹ : 3380, 3250, 1740, 1715, 1660, 1640.

(2) Step 2 ##STR10##

A solution ofD-Lac(OAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Z-(D)-mesoDAP-(L)-GlyOH-(D)-NH₂ (3)(400 mg) in acetic acid (40 ml) was hydrogenated over 10% palladiumcharcoal (100 mg). After removal of the catalyst, the filtrate wasconcentrated under reduced pressure. The residue was dissolved in 50%aqueous methanol (10 ml) and the mixture was stirred for 3 hours at roomtemperature, during which time pH of the mixture was maintained at 9 to10 with 10% aqueous sodium carbonate. After removal of methanol, the theconcentrate was acidified to PH 2 with 1N-hydrochloric acid. Thissolution was put on a column packed with HP 20 (100 ml) and eluted withwater. The elute was concentrated to about 10 ml and lyophilized to giveD-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH-(D)-NH₂ (4) (210 mg).

NMR (D₂ O), δ: 1.38 (d, J=7 Hz, 3H), 1.42 (d, J=7 Hz, 3H), 1.6-2.5 (m,10H), 3.96 (s, 2H), 4.03 (t, J=6 Hz, 1H), 4.2-4.5 (m, 5H),

IR (Nujol) cm⁻⁴ : 3280, 1720 (sh), 1650.

We claim:
 1. A compound of the formula or its pharmaceuticallyacceptable salt: ##STR11## wherein R¹ is heptanoyl, phenylacetyl,2-hydroxypropionyl or 2-acetoxypropionyl,R² is carboxy orbenzyloxycarbonyl, R³ is carboxy, R⁴ is hydrogen, t-butoxycarbonyl orbenzyloxycarbonyl, and R⁵ is hydrogen or methyl.
 2. A compound accordingto claim 1, wherein R¹ is 2-acetoxypropionyl, R² is benzyloxycarbonyl,R³ is carboxy, R⁴ is t-butoxycarbonyl and R⁵ is hydrogen.
 3. A compoundaccording to claim 1, wherein R¹ is 2-hydroxypropionyl, R² and R³ areeach carboxy, R⁴ and R⁵ are each hydrogen.
 4. A compound according toclaim 1, wherein R¹ is heptanoyl, R² is benzyloxycarbonyl, R³ iscarboxy, R⁴ is t-butoxycarbonyl and R⁵ is methyl.
 5. A compoundaccording to claim 1, wherein R¹ is heptanoyl, R² and R³ are eachcarboxy, R⁴ is hydrogen and R⁵ is methyl.
 6. A compound according toclaim 1, wherein R¹ is phenylacetyl, R² is benzyloxycarbonyl, R³ iscarboxy, R⁴ is t-butoxycarbonyl and R⁵ is methyl.
 7. A compoundaccording to claim 1, wherein R¹ is phenylacetyl, R² and R³ are eachcarboxy, R⁴ is hydrogen and R⁵ is methyl.
 8. A compound according toclaim 1, wherein R¹ is 2-acetoxypropionyl, R² is benzyloxycarbonyl, R³is carboxy, R⁴ is benzyloxycarbonyl and R⁵ is hydrogen.